Streptococcus antigen



Patented Apr. 25, 1939 UNITED STATES STBEPTOCOOCUS ANTIGEN George F.Hibbert, Chicago, Ill., assignor to Eli Lilly and Company, Indianapolis,11111., a corporation of Indiana No Drawing. Application April 80, 1938,

Serial No. 205,365

5 Claims. (01. 16'i-78) It is the object of my invention to produce anantigen that is specific in the treatment of (socalled) trichomonasvaginalis vaginitis.

The trichomonas vaginalis has been known for 5 about one hundred years.

1856 (Scanzoni) it has been rather generally accepted that it is thecausative agent of the particular type of vaginitis that is known astrichomonas vaginalis vaginitis.

in But there have been some in recent years who have doubted that thetrichomonas vaginalis was the true causative agent of this disease, andhave even doubted that it was pathogenic at all; although no one priorto my work, so far as I 15 have been able to discover, has establishedwhat the real causative agent is. Thus in 1926 Dobell conducted certainexperiments on monkeys, which tended to establish, and I think didestablish, that the trichomonas vaginalis was not the causa- 20 tiveagent of this type of vaginitis; for he repeatedly infected the vaginaltracts of monkeys with the trichomonas vaginalis, and reported that noneof the symptoms of the so-called trichomonas vaginalis vaginitisresulted.

In a paper by me, published in the American Journal of Obstetrics andGynecology for April, 1933, page 465 et seq., I confirmed-Dobellsobservations, but in human cases. As reported in that paper, I observedmany human cases where the trichomonas vaginalis was present in thevagina, but there were none of the symptoms of trichomonas vaginalisvaginitis. In addition, I deliberately implanted several patients withtrichomonas vaginalis, and obtained a fairly ac- .13 tive growth of itin the vagina; but although that growth persisted for weeks, there wereabsolutely no symptomsof trichomonas vaginalis vaginitis.

These observations are supported by the recent publication of Craig andFaust (1937), who say: 4n Although the trichomonas vaginalis is usuallyfound in an abnormal vagina, and while gynecologists almost universallyregard a form of vaginitis as being caused by the organism, there is noevidence of scientific value demonstrating that it is a pathogenicparasite. On the other hand there is much experimental evidence provingthat it is harmless and incapable of causing any pathological lesions.

In my aforesaid 1933 paper, which indicated that the so-calledtrichomonas vaginalis vaginitis in human beings was almost certainlycaused by some agent other than the trichomonas vaginalis, I suggestedthat some streptococcus probably played a very important part in thatcausation, and that that streptococcus was non-hemolytic.

(Donn, 1837.) Since The discussion that followed the presentation ofthat paper (as that discussion appears on pages 471 to 474 of the April,1933, issue of the American Journal of Obstetrics and Gynecology), madeit evident that other gynecologists'were not then in accord with myviews, and felt that my work as reported at that time did not establisheither that the trichomonas vaginalis was not the cause of thatvaginitis or that a streptococcus was such cause.

As a result of continuous work since that reported in my 1933 paper, Ihave now been able to isolate a specific streptococcus which will causethe so-called trichomonas vaginalis vaginitis. It is the streptococcussubacidus. This is reported in a paper which I read before the CentralAssociation of Obstetrics and Gynecology at its meeting in Dallas,Texas, in October of 1937, and which has been published in the AmericanJournal of Obstetrics and Gynecology for August, 1938, volume 36, N0. 2,pages 219-229. While that streptococcus subacidus had been identifiedearlier as a species of streptococcus (in Holmans classification ofstreptococci),.so far as I know it had never been associated with theso-called trichomonas vaginalis vaginitis; and neither it nor anythingderived from it had been used either to produce or to treat theso-called trichomonas vaginalis vaginitis. Holmans classification ofstreptococci is published in the Journal of Medical Research, volume 34,1916, pages 377-443; and the position of-streptococcus subacidus in thestreptococci group is shown in the chart appearing on page 388 of thatpaper. Table 9 of that paper, on pages 408 and 409, and the discussionof it which begins on page 409, are specifically directed to thestreptococcus subacidus and its identifying characteristics. Theidentifying characteristics of streptococcus subacidus are also given onpage 57 of Bergeys Manual of Determinative Bacteriology, fourth edition,published in 1934.

I have now isolated streptococcus subacidus from the vaginal dischargein human cases of the so-called trichomonas vaginalis vaginitis. I havemade cultures of that isolated streptococcus subacidus. I have implantedthat culture into the vaginal tracts of normal human beingsthose with notrace of any vaginitisfiand have produced the characteristic pathologyof so-called trichomonas vaginalis vaginitis. After the production ofthat characteristic pathology, and its persistence for several weeks, Ihave recovered the streptococcus subacidus from those same individuals,and have isolated and identified it. Thus I have complied with Kochspostulates.

In addition, from that recovered, isolated, and identified streptococcussubacidus, I have pre pared a specific vaccine and a specific antigenicfiltrate, and have used both of them to treat these artificiallyproduced cases (as well as other cases), and have succeeded in renderingthem symptom-free; and they remained symptom-free for periods of months.

Further, I have injected rabbits intravenously with streptococcussubacidus, using repeated and progressively increasing doses, and afterseveral weeks have obtained the blood serum from the rabbits so treated.When that blood serum was tested against streptococcus subacidus,agglutination or clumping occurred; thusdemonstrating that thestreptococcus subacidus isantigenic.

Also, I'have taken bloodse'rumfrom patients who have had theso-calleditrichomonas vaginalis vaginitis, and. who have been renderedsymptomfree by treatment with my specific-filtrate and vaccine obtainedfromkthe; streptococcus subacidus. I have tested that humanblood-serumagainst the streptococcus'subacldus, and have foundithat agglutinationorblumping occurs; thus demonstrating that'l'the streptococcus subacidusis: antigenic: .inlhhumanbeings; and that specific and-bodiesproduced-through. its use are associated withgpatients 'who havelbeenmade,

symtom-free; In isolatin so producedewas s'treaked ion a-steril'eblood-agar plate} and thatpiate was. allowed to incubate forapproximatelytwenty four.-hours.'- Various colomes-growths from singleorganisms-appeared Among these were some that. were hemolytic, asindicated by a definite circular area of hemolysis about the colony; anda stained smear showed J these to-be streptococcii" Contrary towhatmight have been supposed from what was reported in 7 my aforesaid 1933paper, namely that non-hemog lyticstreptococ'ci played a-very importantpart in trichomonas 'vaginalis'vaginitis, I-found that it was these.hemolytic "streptococci which-are the inalis vaginitis. These hemolyticstreptococci were identified as the streptococcus subacidus. 1

This identification showed the following char acterlstics of theso-isolated hemolytic streptococcus. It appears as a gram-positiveslightly elongated small diplococcus, non-capsulated, and develops shortchains both on blood-agar and in broth. In sugar media it fermentsdextrose, sacchrose, maltose, levulose, and trehalose; while in thirteenother sugarsit produced no fermentation. The zone of hemolysis onblood-agar plates is small but definite, although perhaps not sodefinite as some other strains of hemolytic strepto cocci; and no greencoloration was noted. It is killed in ten minutes at a temperature of 65F.

It was this hemolytic streptococcus, thus isolated andidentifled whichwas used in the im-. plantations previously referred to and whichcausedthe symptoms of trichomonas vaginalis vaginitis. I The implantation inthe human patients was as follows:

identifying the streptococcus subacidusi-x'fromf unianacases or so-called trich-r monas vaginalis-yal'gi-nitls; I obtained a [sampleof lthe vaginal {discharge on; ajsterile- ;s'wab,' and im: plantedsterile1-$broth1'with-fthat-samplfif This broth was allowed= "-toiincubate arer {M DI-oximately twentyfoure hours duringfwhlcli'a-growth'of organisms occurred-.*-lThen's'a'dropfromthe culture .1;rate is" the filtrate which'Iuse.

First, a sterile cotton swab which had been saturated with a livingbroth culture of the isolated and identified streptococcus was appliedto the vaginal mucosa. Second, a sterile cotton tampon which had beensaturated with the same culture was placed in the vagina, and allowed toremain there for twenty-four hours. Some of the subjects were normalwomen of child-bearing age, while others had shortly before undergonesupra-pubic hysterectomy. Both groups developed the characteristicsymptoms of so-called trichomonas vaginalis vaginitis; the symptomsproduced in the former group were more severe than those produced in thelatter group.

From this isolated and identified streptococcus, thus shown to be thecausative agent of trichomonas vaginalis vaginitis, I have preparedantigens which are useful in the treatment of that disease. I haveprepared two forms of such antigens, which will be referred to as thefiltrate and the vaccine. These may be prepared as follows:

The isolated and identified streptococcus subacidus is transferred to asterile culture medium, conveniently agar or broth, and incubated underusual incubating conditions for about one day.

Propagation of the organisms is carried out in the usual manner. l

To make the filtrate:

1 The; organisms are transferred to a sterile --growth-promoting liquidmedium, most conveni- .ently.broth, and incubated for about two days,

under usual incubating conditions at about normal blood-temperature,andthenheld at room temperatureforten or-twel ve-days.

n After},thiseprocedure, the culture is passed throughasterilizing'filter, and the-resultant fil- To make the vaccine: j-'.Theorganisms are transferred to l a sterile growth pronioting medium(liquid or solid, such as agar or broth), andincubated for about oneday. If' agar is the, mediumused, the organisms are harvested in normal.saline solution; and if broth 0 is the medium used, the culture iscentrifuged to get the organisms closely compacted. and the suipernatantliquid discarded'and the compacted organisms aretakenup in nomal'salin'esolution. In either event, the organisms in the suspension are thenkilled-in any suitable way, as by heat. The

suspension is then standardized to a definite count, conveniently about250"m'illion per cc. This is the-vaccine which I use.

' Out of abundant caution, itis desirable to add a preservative to boththe filtrate and the vaccine,

. such as 1 part in 10,000 of Merthiolate (sodium ethyl mercurlthiosalicylate, Lilly) {although no preservative is essential, and nonewas used in the experimental .work for establishing the facts -follows:

1. The vagina and external genitalia are wiped free of discharge, withdry sterile cotton pledgets.

2. A sterile tampon, preferably cotton, sat: urated with the filtrate,is inserted-in the vagina,

and removed by the patient'after about twenty four hours. Cotton isbetter for the tampon than is lambs wool, because lambs wool is-notsufilciently absorbent; and I believe that it is better than any otherabsorbent, although of course other absorbent materials, if sufiicientlyabsorbent, may be used. j

3. The filtrate is applied to the external genitalia, as by swabbing,and allowed to dry in place. v

4. At the same time, an injection of the vaccine is given, preferably anintradermal injection and conveniently on the fiexor surface of theforearm.

5. This double treatment, with both filtrate and vaccine, is repeated atintervals, conveniently of about a week, taking care to avoid theperiods of menstruation.

6. For best results I deem it desirable that no other topical treatmentbe made than with the filtrate-as by douches, powders, vaginalsuppositories, or any other antiseptic or germicide.

In administering the vaccine intradermally, it is desirable to startwith a fairly small dose, of the order of about one-twentieth of a cc.Successive doses may be increased or not, depending upon the severity ofthe skin reaction; for I deem it desirable that care be taken that theskin reaction should not be more severe than is sufllcient to produce anarea of redness of about two inches in diameter. Ordinarily the maximumdose should not be above about onehalf of a cc.

In the majority of cases, not more than seven treatments are required tocause the symptoms to disappear. A number of patients so treated havenowbeen symptom-free for periods ranging from six to nine months. Insome instances there has been recurrence, especially followingmenstruation; and in that event it is desirable to repeat the treatmentthrough at least anof cases, by use of the vaccine intradermally in themale as above described for the female, and

by anterior urethral injections of the filtrate.

I claim as my invention:

1. An antigen that is specific in the treatment of so-called trichomonasvaginalis vaginitis, comprising a vaccine consisting substantially ofheat-killed streptococci subacidus.

2. An antigen that is specific in the treatment of so-called trichomonasvaginalis vaginitis, comprising a vaccine consisting substantially ofkilled streptococci subacidus.

3. An antigen that is specific in the treatment of so-called trichomonasvaginalis vaginitis,

comprising a sterile filtrate of a broth culture of streptococcussubacidus.

4. An antigen that is specific in the treatment of so-called trichomonasvaginalis vaginitis, comprising a sterile filtrate of a culture ofstreptococcus subacidus.

5. A sterile antigen from streptococcus subacidus and specific in thetreatment of so-called trichomonas vaginalis vaginitls.-

GEORGE EHIBBERT.

